Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus

Exoenzyme C3stau2 from Staphylococcus aureus is a new member of the family of C3-like ADP-ribosyltransferases that ADP-ribosylates RhoA, -B, and -C. Additionally, it modifies RhoE and Rnd3. Here we report on studies of the structure-function relationship of recombinant C3stau2 by site-directed mutagenesis. Exchange of Glu(180) with leucine caused a complete loss of both ADP-ribosyltransferase and NAD glycohydrolase activity. By contrast, exchange of the glutamine residue two positions upstream (Gln(178)) with lysine blocked ADP-ribosyltransferase activity without major changes in NAD glycohydrolase activity. NAD and substrate binding of this mutant protein was comparable to that of the recombinant wild type. Exchange of amino acid Tyr(175), which is part of the recently described "ADP-ribosylating toxin turn-turn" (ARTT) motif [Han, S., Arvai, A. S., Clancy, S. B., and Tainer, J. A. (2001) J. Mol.Biol. 305, 95-107], with alanine, lysine, or threonine caused a loss of or a decrease in ADP-ribosyltransferase activity but an increase in NAD glycohydrolase activity. Recombinant C3stau2 Tyr175Ala and Tyr175Lys were not precipitated by matrix-bound Rho, supporting a role of Tyr(175) in protein substrate recognition. Exchange of Arg(48) and/or Arg(85) resulted in a 100-fold reduced transferase activity, while the recombinant C3stau2 double mutant R48K/R85K was totally inactive. The data indicate that amino acid residues Arg(48), Arg(85), Tyr(175), Gln(178), and Glu(180) are essential for ADP-ribosyltransferase activity of recombinant C3stau2 and support the role of the ARTT motif in substrate recognition of RhoA by C3-like ADP-ribosyltransferases.     

ADP-ribosylation and functional effects of Pseudomonas exoenzyme S on cellular RalA

Exoenzyme S (ExoS) is a bifunctional virulence factor directly translocated into eukaryotic cells by the type III secretory process of Pseudomonas aeruginosa. Bacterial translocation of ExoS into epithelial cells is associated with diverse effects on cell function, including inhibition of growth, alterations in cell morphology, and effects on adherence processes. Preferred substrates of the ADP-ribosyltransferase (ADPRT) portion of ExoS include low molecular weight G-proteins (LMWG-proteins) in the Ras family. In examining the ADP-ribosylation and functional effects of ExoS on RalA, ExoS was found to ADP-ribosylate endogenous RalA and recombinant RalADeltaCAAX at multiple sites, with Arg52 identified as the preferred site of ADP-ribosylation. The binding of RalA to the Ral binding domain (RBD) of its downstream effector, RalBP1, was inhibited by bacterially translocated ExoS, indicating an effect of ExoS on cellular RalA function. In vitro analyses confirmed that ADP-ribosylation of RalA directly interfered with its ability to bind to the RBD of RalBP1. The studies support the fact that RalA is a cellular substrate of bacterially translocated ExoS and that ADP-ribosylation by ExoS affects RalA interaction with its downstream effector, RalBP1.     

Crystal structure of the Clostridium limosum C3 exoenzyme

C3-like toxins ADP-ribosylate and inactivate Rho GTPases. Seven C3-like ADP-ribosyltransferases produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus were identified and two representatives - C3bot from C. botulinum and C3stau2 from S. aureus - were crystallized. Here we present the 1.8 Å structure of C. limosum C3 transferase C3lim and compare it to the structures of other family members. In contrast to the structure of apo-C3bot, the canonical ADP-ribosylating turn turn motif is observed in a primed conformation, ready for NAD binding. This suggests an impact on the binding mode of NAD and on the transferase reaction. The crystal structure explains why auto-ADP-ribosylation of C3lim at Arg41 interferes with the ADP-ribosyltransferase activity of the toxin.     

Recognition of RhoA by Clostridium botulinum C3 exoenzyme

The C3-like ADP-ribosyltransferases exhibit a very confined substrate specificity compared with other Rho-modifying bacterial toxins; they selectively modify the RhoA, -B, and -C isoforms but not other members of the Rho or Ras subfamilies. In this study, the amino acid residues involved in the RhoA substrate recognition by C3 from Clostridium botulinum are identified by applying mutational analyses of the nonsubstrate Rac. First, the minimum domain responsible for the recognition by C3 was identified as the N-terminal 90 residues. Second, the combination of the N-terminal basic amino acids ((Rho)Arg(5)-Lys(6)), the acid residues (Rho)Glu(47) and (Rho)Glu(54) only slightly increases ADP-ribosylation but fully restores the binding of the respective mutant Rac to C3. Third, the residues (Rho)Glu(40) and (Rho)Val(43) also participate in binding to C3 but they are mainly involved in the correct formation of the ternary complex between Rho, C3, and NAD(+). Thus, these six residues (Arg(5), Lys(6), Glu(40), Val(43), Glu(47), and Glu(54)) distributed over the N-terminal part of Rho are involved in the correct binding of Rho to C3. Mutant Rac harboring these residues shows a kinetic property with regard to ADP-ribosylation, which is identical with that of RhoA. Differences in the conformation of Rho given by the nucleotide occupancy have only minor effects on ADP-ribosylation.     

Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa

The 14-3-3 proteins are a family of conserved, dimeric proteins that interact with a diverse set of ligands, including molecules involved in cell cycle regulation and apoptosis. It is well-established that 14-3-3 binds to many ligands through phosphoserine motifs. Here we characterize the interaction of 14-3-3 with a nonphosphorylated protein ligand, the ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa. By using affinity chromatography and surface plasmon resonance, we show that the zeta isoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro. The interaction between ExoS and 14-3-3zeta is of high affinity, with an equilibrium dissociation constant of 7 nM. ExoS lacks any known 14-3-3 binding motif, but to address the possibility that 14-3-3 binds a noncanonical phosphoserine site, we assayed ExoS for protein-bound phosphate by using mass spectrometry. No detectable phosphoproteins were found. A phosphopeptide ligand of 14-3-3, pS-Raf-259, was capable of inhibiting the binding of 14-3-3 to ExoS, suggesting that phosphorylated and nonphosphorylated ligands may share a common binding site, the conserved amphipathic groove. It is conceivable that 14-3-3 proteins may bind both phosphoserine and nonphosphoserine ligands in cells, possibly allowing kinase-dependent as well as kinase-independent regulation of 14-3-3 binding.     

Immuno-crossreactivity between botulinum neurotoxin type C1 or D and exoenzyme C3

Botulinum neurotoxin type D and exoenzyme C3 have been separately purified from Clostridium botulinum strain D-1873 to apparent homogeneity. Both ADP-ribosylated a rat liver cytosolic protein of 24 kDa. The N-terminal amino acid sequence of C3 was determined and showed a low degree of homology with those of the light and heavy chains of neurotoxins of various types which have been reported previously. However, a polyclonal antibody raised against C3 cross-reacted with the light chains, but not with the heavy chains, of type C1 and D neurotoxins. Furthermore, a monoclonal antibody recognizing the light chains of type C1 and D neurotoxins interacted with C3. These results suggest that the light chain of type C1 or D neurotoxin and exoenzyme C3 share at least one epitope in common with each other.     

NAD binding induces conformational changes in Rho ADP-ribosylating clostridium botulinum C3 exoenzyme

We have solved the crystal structures of Clostridium botulinum C3 exoenzyme free and complexed to NAD in the same crystal form, at 2.7 and 1.95 A, respectively. The asymmetric unit contains four molecules, which, in the free form, share the same conformation. Upon NAD binding, C3 underwent various conformational changes, whose amplitudes were differentially limited in the four molecules of the crystal unit. A major rearrangement concerns the loop that contains the functionally important ARTT motif (ADP-ribosyltransferase toxin turn-turn). The ARTT loop undergoes an ample swinging motion to adopt a conformation that covers the nicotinamide moiety of NAD. In particular, Gln-212, which belongs to the ARTT motif, flips over from a solvent-exposed environment to a buried conformation in the NAD binding pocket. Mutational experiments showed that Gln-212 is neither involved in NAD binding nor in the NAD-glycohydrolase activity of C3, whereas it plays a critical role in the ADP-ribosyl transfer to the substrate Rho. We observed additional NAD-induced movements, including a crab-claw motion of a subdomain that closes the NAD binding pocket. The data emphasized a remarkable NAD-induced plasticity of the C3 binding pocket and suggest that the NAD-induced ARTT loop conformation may be favored by the C3-NAD complex to bind to the substrate Rho. Our structural observations, together with a number of mutational experiments suggest that the mechanisms of Rho ADP-ribosylation by C3-NAD may be more complex than initially anticipated.     

Purification and characterization of ADP-ribosyltransferases (exoenzyme C3) of Clostridium botulinum type C and D strains

By cation-exchange column chromatography followed by gel filtration or hydroxylapatite column chromatography, ADP-ribosyltransferases (exoenzyme C3) were isolated from culture supernatants of Clostridium botulinum type C strains Stockholm (CST) and 6813 (C6813) and from type D strains South African (DSA) and 1873 (D1873), and their molecular properties were compared. The purified C3 enzymes were homogeneous in polyacrylamide gel electrophoresis. The C3 enzymes existed as single-chain polypeptides with molecular masses of 25.0 to 25.5 kDa and transferred ADP-riboses to the same substrates in rat brain membrane extract. The C3 enzymes could be roughly classified into two groups with respect to amino acid composition, amino-terminal sequence, and antigenicity. One group contains the C3 enzymes of strains C6813 and DSA, and the other contains those of strains CST and D1873. The specific activity of the C3 enzyme of strain C6813 was about 15 times higher than that of the C3 enzyme of strain CST. These results indicate that the classification of the C3 molecules differs from that of the neurotoxin molecules.     

Crystal structure of the C3bot-RalA complex reveals a novel type of action of a bacterial exoenzyme

C3 exoenzymes from bacterial pathogens ADP-ribosylate and inactivate low-molecular-mass GTPases of the Rho subfamily. Ral, a Ras subfamily GTPase, binds the C3 exoenzymes from Clostridium botulinum and C. limosum with high affinity without being a substrate for ADP ribosylation. In the complex, the ADP-ribosyltransferase activity of C3 is blocked, while binding of NAD and NAD-glycohydrolase activity remain. Here we report the crystal structure of C3 from C. botulinum in a complex with GDP-bound RalA at 1.8 A resolution. C3 binds RalA with a helix-loop-helix motif that is adjacent to the active site. A quaternary complex with NAD suggests a mode for ADP-ribosyltransferase inhibition. Interaction of C3 with RalA occurs at a unique interface formed by the switch-II region, helix alpha3 and the P loop of the GTPase. C3-binding stabilizes the GDP-bound conformation of RalA and blocks nucleotide release. Our data indicate that C. botulinum exoenzyme C3 is a single-domain toxin with bifunctional properties targeting Rho GTPases by ADP ribosylation and Ral by a guanine nucleotide dissociation inhibitor-like effect, which blocks nucleotide exchange.     

Entrapment of Rho ADP-ribosylated by Clostridium botulinum C3 exoenzyme in the Rho-guanine nucleotide dissociation inhibitor-1 complex

RhoA, -B, and -C are ADP-ribosylated by Clostridium botulinum exoenzyme C3 to induce redistribution of the actin filaments in intact cells, a finding that has led to the notion that the ADP-ribosylation blocks coupling of Rho to the downstream effectors. ADP-ribosylation, however, does not alter nucleotide binding, intrinsic, and GTPase-activating protein-stimulated GTPase activity. ADP-ribosylated Rho is even capable of activating the effector protein ROK in a recombinant system. Treatment of cells with a cell-permeable chimeric C3 toxin led to complete localization of modified Rho to the cytosolic fraction based on the complexation of ADP-ribosylated Rho with the guanine-nucleotide dissociation inhibitor-1 (GDI-1). The modified complex turned out to be resistant to phosphatidylinositol 4,5-bisphosphate- and GTPgammaS-induced release of Rho from GDI-1. Thus, ADP-ribosylation leads to entrapment of Rho in the GDI-1 complex. The increased stability of the GDI complex prevented binding of Rho to membrane-associated players of the GTPase cycle such as the activating guanine nucleotide exchange factors and effector proteins.     

Probing the action of Clostridium toxins B and exoenzyme C3 for detection of Rho-like motifs of alfalfa proteins

Small GTP-binding Rho proteins are involved in signalling, cell polarity, membrane outgrowths and actin stabilization in eukaryotes. Known plant homologues represent essentially the Rac subfamily and an original Rop (Rho in pollen). Mammalian Rho proteins are preferential targets of clostridial toxins. In alfalfa (Medicago sativa L.) cells, Clostridium botulinum C3-exoenzyme (C3) provoked disassembly of the actin cytoskeleton, similar to its effect in mammalian cells. In alfalfa proteins, several epitopes appear to be recognized by commercial antibodies raised against peptides characteristic for human Rho. One ≈ 40-kDa band was detected immunologically by anti-RhoB: a protein of this size was ADP-ribosylated by C3 and glucosylated in vitro by Clostridium difficile toxin B, without interference between the two nor from phosphatidyl inositide. C3 was also active upon a 34-kDa band which contained protein(s) immunoreactive with anti-Rac2 and which bound [γ³⁵S]-GTP, but was glucosylated by neither toxin B nor Clostridium sordellii Lethal Toxin. An 18-kDa band detected by [γ³⁵S]-GTP overlay was immunologically recognized by anti-Rac1. Anti-Cdc42Hs recognized a 54-kDa band. Substrates to toxin B and C3 were purified from alfalfa cell culture and partially sequenced: they included two proteins, P40 and P41, of ≈ 40 kDa (by SDS-electrophoresis). P40 appears to constitute a tetrameric aldolase (160 kDa by gel filtration; EC 4.1.2.13) whose activity is partially inhibited by toxin B and the anti-RhoB.     

Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.

Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot. Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein. When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function. NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins. When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins. Nevertheless, p21.bot was immunologically distinct from p21ras.     

ADP-ribosylation by Clostridium botulinum C3 exoenzyme increases steady-state GTPase activities of recombinant rhoA and rhoB proteins.

ADP-ribosylation of recombinant rhoA and rhoB proteins by Clostridium botulinum C3 exoenzyme increased steady-state GTP hydrolysis by 50 to 80%. ADP-ribosylation and increase in GTP hydrolysis occurred at similar concentrations of C3, depended on the presence of NAD and were prevented by anti-C3 antibody or heat inactivation of C3. In contrast, GTP hydrolysis by Ile-41 rhoA or Ha-ras, which are no substrates for the transferase, were not affected by C3. ADP-ribosylation facilitated the [3H]GDP release and subsequently, the binding of [3H]GTP to rhoA. The data indicate that the increase in the steady-state GTPase activity by ADP-ribosylation is caused by increasing the rate of GDP release which is suggested to be the rate limiting step of the GTPase cycle of the small GTP-binding proteins.     

Exchange of glutamine-217 to glutamate of Clostridium limosum exoenzyme C3 turns the asparagine-specific ADP-ribosyltransferase into an arginine-modifying enzyme

C3-like ADP-ribosyltransferaseses are produced by Clostridium species, Bacillus cereus, and various Staphylococcus aureus strains. The exoenzymes modify the low-molecular-mass GTPases RhoA, B, and C. In structural studies of C3-like exoenzymes, an ARTT-motif (ADP-ribosylating turn-turn motif) was identified that appears to be involved in substrate specificity and recognition (Han, S., Arvai, A. S., Clancy, S. B., Tainer, J. A. (2001) J. Mol. Biol. 305, 95-107). Exchange of Gln217, which is a key residue of the ARTT-motif, to Glu in C3 from Clostridium limosum results in inhibition of ADP-ribosyltransferase activity toward RhoA. The mutant protein is still capable of NAD-binding and possesses NAD+ glycohydrolase activity. Whereas recombinant wild-type C3 modifies Rho proteins specifically at an asparagine residue (Asn41), Gln217Glu-C3 is capable of ADP-ribosylation of poly-arginine but not poly-asparagine. Soybean trypsin inhibitor, a model substrate for many arginine-specific ADP-ribosyltransferases, is modified by the Gln217Glu-C3 transferase. Also in C3 ADP-ribosyltransferases from Clostridium botulinum and B. cereus, the exchange of the equivalent Gln residue to Glu blocked asparagine modification of RhoA but elicited arginine-specific ADP-ribosylation. Moreover, the Gln217Glu-C3lim transferase was able to ADP-ribosylate recombinant wild-type C3lim at Arg86, resulting in decrease in ADP-ribosyltransferase activity of the wild-type enzyme. The data indicate that the exchange of one amino acid residue in the ARTT-motif turns the asparagine-modifying ADP-ribosyltransferases of the C3 family into arginine-ADP-ribosylating transferases.     

Delineation of exoenzyme S residues that mediate the interaction with 14-3-3 and its biological activity

14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells, which play an important role in a multitude of signaling pathways. 14-3-3 proteins bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner. More than 200 14-3-3 binding partners have been found that are involved in cell cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. A phosphorylation-independent interaction has been reported to occur between 14-3-3 and a C-terminal domain within exoenzyme S (ExoS), a bacterial ADP-ribosyltransferase toxin from Pseudomonas aeruginosa. In this study, we have investigated the effect of amino acid mutations in this C-terminal domain of ExoS on ADP-ribosyltransferase activity and the 14-3-3 interaction. Our results suggest that leucine-428 of ExoS is the most critical residue for ExoS enzymatic activity, as cytotoxicity analysis reveals that substitution of this leucine significantly weakens the ability of ExoS to mediate cell death. Leucine-428 is also required for the ability of ExoS to modify the eukaryotic endogenous target Ras. Finally, single amino acid substitutions of positions 426-428 reduce the interaction potential of 14-3-3 with ExoS in vitro.     

Purification of plasma membrane penicillinase from Bacillus licheniformis 749/C and comparison with exoenzyme.

The membrane penicillinase of Bacillus licheniformis 749/C has been demonstrated to be a phospholipoprotein. The homogeneous enzyme gives a positive reaction for phosphorous and for unsaturated fatty acids, has a molecular weight of 33,000 in contrast to 29,000 for the exoenzyme, and contains 8 to 9 additional residues of aspartate or asparagine, 4 to 5 of serine, 7 of glutamate or glutamine, and 4 to 5 of glycine per mole. The COOH-terminal sequence of both membrane and exoenzymes is -Met-Asn-Gln-Lys-COOH; hence the extra peptide portion present in the membrane enzyme is not attached to the COOH-terminus of the exoenzyme. Procedures which readily detected the lysine residue at the NH2 terminus of the exoenzyme did not yield a positive test with the membrane form. The NH2 terminus of the membrane enzyme may be blocked by or linked to the phospholipid. A procedure for the preparation of membrane penicillinase on a large scale and an improved method for purification of the exoenzyme have been developed.